AUTACs: Cargo-Specific Degraders Using Selective Autophagy.

Protein silencing represents an essential tool in biomedical research. Targeted protein degradation (TPD) strategies exemplified by PROTACs are rapidly emerging as modalities in drug discovery. However, the scope of current TPD techniques is limited because many intracellular materials are not substrates of proteasomal clearance. Here, we described a novel targeted-clearance strategy (autophagy-targeting chimera [AUTAC]) that contains a degradation tag (guanine derivatives) and a warhead to provide target specificity. As expected from the substrate scope of autophagy, AUTAC degraded fragmented mitochondria as well as proteins. Mitochondria-targeted AUTAC accelerated both the removal of dysfunctional fragmented mitochondria and the biogenesis of functionally normal mitochondria in patient-derived fibroblast cells. Cytoprotective effects against acute mitochondrial injuries were also seen. Canonical autophagy is viewed as a nonselective bulk decomposition system, and none of the available autophagy-inducing agents exhibit useful cargo selectivity. With its target specificity, AUTAC provides a new modality for research on autophagy-based drugs.

Regulation of innate immune and inflammatory responses by supersulfides.

Innate immunity plays an important role in host defense against microbial infections. It also participates in activation of acquired immunity through cytokine production and antigen presentation. Pattern recognition receptors such as Toll-like receptors and nucleotide oligomerization domain-like receptors sense invading pathogens and associated tissue injury, after which inflammatory mediators such as pro-inflammatory cytokines and nitric oxide are induced. Supersulfides are molecular species possessing catenated sulfur atoms such as persulfide and polysulfide moieties. They have recently been recognized as important regulators in cellular redox homeostasis by acting as potent antioxidants and nucleophiles. In addition, recent studies suggested that supersulfides are critically involved in the regulation of innate immune and inflammatory responses. In this review, we summarize current knowledge of the chemistry and biology of supersulfides, with particular attention to their roles in regulation of innate immune, and inflammatory responses. Studies with animal models of infection and inflammation demonstrated the potent anti-inflammatory functions of supersulfides such as blocking pro-inflammatory signaling cascades, reducing oxidative stresses, and inhibiting replication of microbial pathogens including severe acute respiratory syndrome coronavirus 2. Precise understanding of how supersulfides regulate innate immune responses is the necessary requirement for developing supersulfide-based diagnostic as well as therapeutic strategies against inflammatory disorders.

Quantitative profiling of supersulfides naturally occurring in dietary meats and beans.

Sulfur is essential in the inception of life and crucial for maintaining human health. This mineral is primarily supplied through the intake of proteins and is used for synthesizing various sulfur-containing biomolecules. Recent research has highlighted the biological significance of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiol and proteins. Ingestion of exogenous sulfur compounds is essential for endogenous supersulfide production. However, the content and composition of supersulfides in foods remain unclear. This study investigated the supersulfide profiles of protein-rich foods, including edible animal meat and beans. Quantification of the supersulfide content revealed that natto, chicken liver, and bean sprouts contained abundant supersulfides. In general, the supersulfide content in beans and their derivatives was higher than that in animal meat. The highest proportion (2.15 %) was detected in natto, a traditional Japanese fermented soybean dish. These results suggest that the abundance of supersulfides, especially in foods like natto and bean sprouts, may contribute to their health-promoting properties. Our findings may have significant biological implications and warrant developing novel dietary intervention for the human health-promoting effects of dietary supersulfides abundantly present in protein-rich foods such as natto and bean sprouts.

Supersulfide catabolism participates in maladaptive remodeling of cardiac cells.

The atrophic myocardium resulting from mechanical unloading and nutritional deprivation is considered crucial as maladaptive remodeling directly associated with heart failure, as well as interstitial fibrosis. Conversely, myocardial hypertrophy resulting from hemodynamic loading is perceived as compensatory stress adaptation. We previously reported the abundant presence of highly redox-active polysulfide molecules, termed supersulfide, with two or more sulfur atoms catenated in normal hearts, and the supersulfide catabolism in pathologic hearts after myocardial infarction correlated with worsened prognosis of heart failure. However, the impact of supersulfide on myocardial remodeling remains unclear. Here, we investigated the involvement of supersulfide metabolism in cardiomyocyte remodeling, using a model of adenosine 5'-triphosphate (ATP) receptor-stimulated atrophy and endothelin-1 receptor-stimulated hypertrophy in neonatal rat cardiomyocytes. Results revealed contrasting changes in intracellular supersulfide and its catabolite, hydrogen sulfide (H2S), between cardiomyocyte atrophy and hypertrophy. Stimulation of cardiomyocytes with ATP decreased supersulfide activity, while H2S accumulation itself did not affect cardiomyocyte atrophy. This supersulfide catabolism was also involved in myofibroblast formation of neonatal rat cardiac fibroblasts. Thus, unraveling supersulfide metabolism during myocardial remodeling may lead to the development of novel therapeutic strategies to improve heart failure.

Reprogrammed transsulfuration promotes basal-like breast tumor progression via realigning cellular cysteine persulfidation.

Basal-like breast cancer (BLBC) is the most aggressive subtype of breast tumors with poor prognosis and limited molecular-targeted therapy options. We show that BLBC cells have a high Cys demand and reprogrammed Cys metabolism. Patient-derived BLBC tumors from four different cohorts exhibited elevated expression of the transsulfuration enzyme cystathione ß-synthetase (CBS). CBS silencing (shCBS) made BLBC cells less invasive, proliferate slower, more vulnerable to oxidative stress and cystine (CySSCy) deprivation, prone to ferroptosis, and less responsive to HIF1-α activation under hypoxia. shCBS xenograft tumors grew slower than controls and exhibited impaired angiogenesis and larger necrotic areas. Sulfur metabolite profiling suggested that realigned sulfide/persulfide-inducing functions of CBS are important in BLBC tumor progression. Supporting this, the exclusion of serine, a substrate of CBS for producing Cys but not for producing sulfide/persulfide, did not exacerbate CySSCy deprivation-induced ferroptosis in shCBS BLBC cells. Impaired Tyr phosphorylation was detected in shCBS cells and xenografts, likely due to persulfidation-inhibited phosphatase functions. Overexpression of cystathione γ-lyase (CSE), which can also contribute to cellular sulfide/persulfide production, compensated for the loss of CBS activities, and treatment of shCBS xenografts with a CSE inhibitor further blocked tumor growth. Glutathione and protein-Cys levels were not diminished in shCBS cells or xenografts, but levels of Cys persulfidation and the persulfide-catabolizing enzyme ETHE1 were suppressed. Finally, expression of enzymes of the oxidizing Cys catabolism pathway was diminished, but expression of the persulfide-producing CARS2 was elevated in human BLBC tumors. Hence, the persulfide-producing pathways are major targetable determinants of BLBC pathology that could be therapeutically exploited.

GRIM-19 is a target of mycobacterial Zn2+ metalloprotease 1 and indispensable for NLRP3 inflammasome activation.

Tuberculosis is a communicable disease caused by Mycobacterium tuberculosis which primarily infects macrophages and establishes intracellular parasitism. A mycobacterial virulence factor Zn2+ metalloprotease 1 (Zmp1) is known to suppress interleukin (IL)-1ß production by inhibiting caspase-1 resulting in phagosome maturation arrest. However, the molecular mechanism of caspase-1 inhibition by Zmp1 is still elusive. Here, we identified GRIM-19 (also known as NDUFA13), an essential subunit of mitochondrial respiratory chain complex I, as a novel Zmp1-binding protein. Using the CRISPR/Cas9 system, we generated GRIM-19 knockout murine macrophage cell line J774.1 and found that GRIM-19 is essential for IL-1ß production during mycobacterial infection as well as in response to NLRP3 inflammasome-activating stimuli such as extracellular ATP or nigericin. We also found that GRIM-19 is required for the generation of mitochondrial reactive oxygen species and NLRP3-dependent activation of caspase-1. Loss of GRIM-19 or forced expression of Zmp1 resulted in a decrease in mitochondrial membrane potential. Our study revealed a previously unrecognized role of GRIM-19 as an essential regulator of NLRP3 inflammasome and a molecular mechanism underlying Zmp1-mediated suppression of IL-1ß production during mycobacterial infection.

Echinochrome Prevents Sulfide Catabolism-Associated Chronic Heart Failure after Myocardial Infarction in Mice.

Abnormal sulfide catabolism, especially the accumulation of hydrogen sulfide (H2S) during hypoxic or inflammatory stresses, is a major cause of redox imbalance-associated cardiac dysfunction. Polyhydroxynaphtoquinone echinochrome A (Ech-A), a natural pigment of marine origin found in the shells and needles of many species of sea urchins, is a potent antioxidant and inhibits acute myocardial ferroptosis after ischemia/reperfusion, but the chronic effect of Ech-A on heart failure is unknown. Reactive sulfur species (RSS), which include catenated sulfur atoms, have been revealed as true biomolecules with high redox reactivity required for intracellular energy metabolism and signal transduction. Here, we report that continuous intraperitoneal administration of Ech-A (2.0 mg/kg/day) prevents RSS catabolism-associated chronic heart failure after myocardial infarction (MI) in mice. Ech-A prevented left ventricular (LV) systolic dysfunction and structural remodeling after MI. Fluorescence imaging revealed that intracellular RSS level was reduced after MI, while H2S/HS- level was increased in LV myocardium, which was attenuated by Ech-A. This result indicates that Ech-A suppresses RSS catabolism to H2S/HS- in LV myocardium after MI. In addition, Ech-A reduced oxidative stress formation by MI. Ech-A suppressed RSS catabolism caused by hypoxia in neonatal rat cardiomyocytes and human iPS cell-derived cardiomyocytes. Ech-A also suppressed RSS catabolism caused by lipopolysaccharide stimulation in macrophages. Thus, Ech-A has the potential to improve chronic heart failure after MI, in part by preventing sulfide catabolism.

Presence of Helicobacter cinaedi in Atherosclerotic Abdominal Aortic Aneurysmal Wall.

Recently, the relationship between Helicobacter cinaedi (H. cinaedi) infection and several diseases, including cardiovascular and central nervous system disorders, bone and soft tissue disorders, and infectious abdominal aortic aneurysms (AAAs), has been reported. Moreover, H. cinaedi may be associated with arteriosclerosis. In the present study, we investigated the association between H. cinaedi infection and clinically uninfected AAAs. Genetic detection of H. cinaedi in the abdominal aneurysm wall was attempted in 39 patients with AAA undergoing elective open surgery between June 2019 and June 2020. DNA samples extracted from the arterial wall obtained during surgery were analyzed using nested polymerase chain reaction (PCR). The target gene region was the H. cinaedi-specific cytolethal distending toxin subunit B (cdtB). Nine (23.1%) of 39 patients showed positive bands corresponding to H. cinaedi, and further sequencing analyses demonstrated the presence of H. cinaedi DNAs in their aneurysm walls. In contrast, all the non-aneurysm arterial walls in our patients were negative for H. cinaedi. In conclusion, this is the first report of the detection of H. cinaedi in the walls of a clinically non-infectious AAA.

Cystathionine γ-Lyase Self-Inactivates by Polysulfidation during Cystine Metabolism.

Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. It also has ß-lyase activity toward cystine, generating cysteine persulfide (Cys-SSH). The chemical reactivity of Cys-SSH is thought to be involved in the catalytic activity of particular proteins via protein polysulfidation, the formation of -S-(S)n-H on their reactive cysteine residues. The Cys136/171 residues of CSE have been proposed to be redox-sensitive residues. Herein, we investigated whether CSE polysulfidation occurs at Cys136/171 during cystine metabolism. Transfection of wild-type CSE into COS-7 cells resulted in increased intracellular Cys-SSH production, which was significantly increased when Cys136Val or Cys136/171Val CSE mutants were transfected, instead of the wild-type enzyme. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CSE polysulfidation occurs at Cys136 during cystine metabolism. In vitro incubation of CSE with CSE-enzymatically synthesized Cys-SSH resulted in the inhibition of Cys-SSH production. In contrast, the mutant CSEs (Cys136Val and Cys136/171Val) proved resistant to inhibition. The Cys-SSH-producing CSE activity of Cys136/171Val CSE was higher than that of the wild-type enzyme. Meanwhile, the cysteine-producing CSE activity of this mutant was equivalent to that of the wild-type enzyme. It is assumed that Cys-SSH-producing CSE activity could be auto-inactivated via the polysulfidation of the enzyme during cystine metabolism. Thus, the polysulfidation of CSE at the Cys136 residue may be an integral feature of cystine metabolism, which functions to down-regulate Cys-SSH synthesis by the enzyme.

Regulation of nitric oxide/reactive oxygen species redox signaling by nNOS splicing variants.

We previously demonstrated different expression patterns of the neuronal nitric oxide synthase (nNOS) splicing variants, nNOS-µ and nNOS-α, in the rat brain; however, their exact functions have not been fully elucidated. In this study, we compared the enzymatic activities of nNOS-µ and nNOS-α and investigated intracellular redox signaling in nNOS-expressing PC12 cells, stimulated with a neurotoxicant, 1-methyl-4-phenylpyridinium ion (MPP+), to enhance the nNOS uncoupling reaction. Using in vitro studies, we show that nNOS-µ produced nitric oxide (NO), as did nNOS-α, in the presence of tetrahydrobiopterin (BH4), an important cofactor for the enzymatic activity. However, nNOS-µ generated more NO and less superoxide than nNOS-α in the absence of BH4. MPP + treatment induced more reactive oxygen species (ROS) production in nNOS-α-expressing PC12 cells than in those expressing nNOS-µ, which correlated with the intracellular production of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a downstream messenger of nNOS redox signaling, and apoptosis in these cells. Furthermore, post-treatment with 8-nitro-cGMP aggravated MPP+-induced cytotoxicity via activation of the H-Ras/extracellular signal-regulated kinase signaling pathway. In conclusion, our results provide strong evidence that nNOS-µ exhibits distinctive enzymatic properties of NO/ROS production, contributing to the regulation of intracellular redox signaling, including the downstream production of 8-nitro-cGMP.

Loss of cell wall integrity genes cpxA and mrcB causes flocculation in Escherichia coli.

Flocculation has been recognized for hundreds of years as an important phenomenon in brewing and wastewater treatment. However, the underlying molecular mechanisms remain elusive. The lack of a distinct phenotype to differentiate between slow-growing mutants and floc-forming mutants prevents the isolation of floc-related gene by conventional mutant screening. To overcome this, we performed a two-step Escherichia coli mutant screen. The initial screen of E. coli for mutants conferring floc production during high salt treatment yielded a mutant containing point mutations in 61 genes. The following screen of the corresponding single-gene mutants identified two genes, mrcB, encoding a peptidoglycan-synthesizing enzyme and cpxA, encoding a histidine kinase of a two-component signal transduction system that contributed to salt tolerance and flocculation prevention. Both single mutants formed flocs during high salt shock, these flocs contained cytosolic proteins. ΔcpxA exhibited decreased growth with increasing floc production and addition of magnesium to ΔcpxA suppressed floc production effectively. In contrast, the growth of ΔmrcB was inconsistent under high salt conditions. In both strains, flocculation was accompanied by the release of membrane vesicles containing inner and outer membrane proteins. Of 25 histidine kinase mutants tested, ΔcpxA produced the highest amount of proteins in floc. Expression of cpxP was up-regulated by high salt in ΔcpxA, suggesting that high salinity and activation of CpxR might promote floc formation. The finding that ΔmrcB or ΔcpxA conferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation in E. coli.

Virucidal effect of monogalactosyl diacylglyceride from a green microalga, Coccomyxa sp. KJ, against clinical isolates of SARS-CoV-2 as assessed by a plaque assay.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and is capable of human-to-human transmission and rapid global spread. Thus, the establishment of high-quality viral detection and quantification methods, and the development of anti-SARS-CoV-2 agents are critical. METHODS: Here, we present the rapid detection of infectious SARS-CoV-2 particles using a plaque assay with 0.5% agarose-ME (Medium Electroosmosis) as an overlay medium. RESULTS: The plaques were capable of detecting the virus within 36-40 h post-infection. In addition, we showed that a monogalactosyl diacylglyceride isolated from a microalga (Coccomyxa sp. KJ) could inactivate the clinical isolates of SARS-CoV-2 in a time- and concentration-dependent manner. CONCLUSIONS: These results would allow rapid quantification of the infectious virus titers and help develop more potent virucidal agents against SARS-CoV-2.

Oxidation of PKGIα mediates an endogenous adaptation to pulmonary hypertension.

Chronic hypoxia causes pulmonary hypertension (PH), vascular remodeling, right ventricular (RV) hypertrophy, and cardiac failure. Protein kinase G Iα (PKGIα) is susceptible to oxidation, forming an interprotein disulfide homodimer associated with kinase targeting involved in vasodilation. Here we report increased disulfide PKGIα in pulmonary arteries from mice with hypoxic PH or lungs from patients with pulmonary arterial hypertension. This oxidation is likely caused by oxidants derived from NADPH oxidase-4, superoxide dismutase 3, and cystathionine γ-lyase, enzymes that were concomitantly increased in these samples. Indeed, products that may arise from these enzymes, including hydrogen peroxide, glutathione disulfide, and protein-bound persulfides, were increased in the plasma of hypoxic mice. Furthermore, low-molecular-weight hydropersulfides, which can serve as "superreductants" were attenuated in hypoxic tissues, consistent with systemic oxidative stress and the oxidation of PKGIα observed. Inhibiting cystathionine γ-lyase resulted in decreased hypoxia-induced disulfide PKGIα and more severe PH phenotype in wild-type mice, but not in Cys42Ser PKGIα knock-in (KI) mice that are resistant to oxidation. In addition, KI mice also developed potentiated PH during hypoxia alone. Thus, oxidation of PKGIα is an adaptive mechanism that limits PH, a concept further supported by polysulfide treatment abrogating hypoxia-induced RV hypertrophy in wild-type, but not in the KI, mice. Unbiased transcriptomic analysis of hypoxic lungs before structural remodeling identified up-regulation of endothelial-to-mesenchymal transition pathways in the KI compared with wild-type mice. Thus, disulfide PKGIα is an intrinsic adaptive mechanism that attenuates PH progression not only by promoting vasodilation but also by limiting maladaptive growth and fibrosis signaling.

8-Nitro-cGMP suppresses mineralization by mouse osteoblasts.

Nitric oxide and reactive oxygen species regulate bone remodeling, which occurs via bone formation and resorption by osteoblasts and osteoclasts, respectively. Recently, we found that 8-nitro-cGMP, a second messenger of nitric oxide and reactive oxygen species, promotes osteoclastogenesis. Here, we investigated the formation and function of 8-nitro-cGMP in osteoblasts. Mouse calvarial osteoblasts were found to produce 8-nitro-cGMP, which was augmented by tumor necrosis factor-α (10 ng/ml) and interleukin-1ß (1 ng/ml). These cytokines suppressed osteoblastic differentiation in a NO synthase activity-dependent manner. Exogenous 8-nitro-cGMP (30 µmol/L) suppressed expression of osteoblastic phenotypes, including mineralization, in clear contrast to the enhancement of mineralization by osteoblasts induced by 8-bromo-cGMP, a cell membrane-permeable analog of cGMP. It is known that reactive sulfur species denitrates and degrades 8-nitro-cGMP. Mitochondrial cysteinyl-tRNA synthetase plays a crucial role in the endogenous production of RSS. The expression of osteoblastic phenotypes was suppressed by not only exogenous 8-nitro-cGMP but also by silencing of the Cars2 gene, indicating a role of endogenous 8-nitro-cGMP in suppressing the expression of osteoblastic phenotypes. These results suggest that 8-nitro-cGMP is a negative regulator of osteoblastic differentiation.

Methods in sulfide and persulfide research.

Sulfides and persulfides/polysulfides (R-Sn-R', n > 2; R-Sn-H, n > 1) are endogenously produced metabolites that are abundant in mammalian and human cells and tissues. The most typical persulfides that are widely distributed among different organisms include various reactive persulfides-low-molecular-weight thiol compounds such as cysteine hydropersulfide, glutathione hydropersulfide, and glutathione trisulfide as well as protein-bound thiols. These species are generally more redox-active than are other simple thiols and disulfides. Although hydrogen sulfide (H2S) has been suggested for years to be a small signaling molecule, it is intimately linked biochemically to persulfides and may actually be more relevant as a marker of functionally active persulfides. Reactive persulfides can act as powerful antioxidants and redox signaling species and are involved in energy metabolism. Recent evidence revealed that cysteinyl-tRNA synthetases (CARSs) act as the principal cysteine persulfide synthases in mammals and contribute significantly to endogenous persulfide/polysulfide production, in addition to being associated with a battery of enzymes including cystathionine ß-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, which have been described as H2S-producing enzymes. The reactive sulfur metabolites including persulfides/polysulfides derived from CARS2, a mitochondrial isoform of CARS, also mediate not only mitochondrial biogenesis and bioenergetics but also anti-inflammatory and immunomodulatory functions. The physiological roles of persulfides, their biosynthetic pathways, and their pathophysiology in various diseases are not fully understood, however. Developing basic and high precision techniques and methods for the detection, characterization, and quantitation of sulfides and persulfides is therefore of great importance so as to thoroughly understand and clarify the exact functions and roles of these species in cells and in vivo.

Endogenous nitrated nucleotide is a key mediator of autophagy and innate defense against bacteria.

Autophagy is a cellular self-catabolic process wherein organelles, macromolecules, and invading microbes are sequestered in autophagosomes that fuse with lysosomes. In this study, we uncover the role of nitric oxide (NO) as a signaling molecule for autophagy induction via its downstream mediator, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP). We found that 8-nitro-cGMP-induced autophagy is mediated by Lys63-linked polyubiquitination and that endogenous 8-nitro-cGMP promotes autophagic exclusion of invading group A Streptococcus (GAS) from cells. 8-nitro-cGMP can modify Cys residues by S-guanylation of proteins. We showed that intracellular GAS is modified with S-guanylation extensively in autophagosomes-like vacuoles, suggesting the role of S-guanylation as a marker for selective autophagic degradation. This finding is supported by the fact that S-guanylated bacteria were selectively marked with polyubiquitin, a known molecular tag for selective transport to autophagosomes. These results collectively indicate that 8-nitro-cGMP plays a crucial role in cytoprotection during bacterial infections or inflammations via autophagy upregulation.

Antioxidative and anti-inflammatory actions of reactive cysteine persulfides.

Cysteine persulfide (CysSSH) and polysulfides (CysS[S] n H, n>1) are cysteine derivatives having sulfane sulfur atoms bound to cysteine thiol. Recent advances in the development of analytical methods for detection and quantification of persulfides and polysulfides have revealed the biological presence, in both prokaryotes and eukaryotes, of persulfide/polysulfide in diverse forms such as CysSSH, glutathione persulfide and protein persulfides. Accumulating evidence has suggested that persulfide/polysulfide species may involve in a variety of biological events such as biosyntheses of sulfur-containing molecules, tRNA modification, regulation of redox-dependent signal transduction, mitochondrial energy metabolism via sulfur respiration, cytoprotection from oxidative stress via their antioxidant activities, and anti-inflammation against Toll-like receptor-mediated inflammatory responses. Development of chemical sulfur donors may facilitate further understanding of physiological and pathophysiological roles of persulfide/polysulfide species, including regulatory roles of these species in immune responses.

Mitochondrial cysteinyl-tRNA synthetase is expressed via alternative transcriptional initiation regulated by energy metabolism in yeast cells.

Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5' complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex-dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast.

8-Nitro-cGMP modulates exocytosis in adrenal chromaffin cells.

Nitric oxide (NO)-mediated production of cyclic guanosine 3',5'-monophosphate (cGMP) is a crucial signaling pathway that controls a wide array of neuronal functions, including exocytotic neurotransmitter release. A novel nitrated derivative of cGMP, 8-nitro-cGMP, not only activates cGMP-dependent protein kinase (PKG), but also has membrane permeability and redox activity to produce superoxide and S-guanylated protein. To date, no studies have addressed the effects of 8-nitro-cGMP on exocytotic kinetics. Here, we aimed to assess the 8-nitro-cGMP-mediated modulation of the depolarization-evoked catecholamine release from bovine chromaffin cells. 8-Nitro-cGMP was produced in bovine chromaffin cells dependent on NO donor. Amperometric analysis revealed that 8-nitro-cGMP modulated the kinetic parameters of secretory spikes from chromaffin cells, particularly decreased the speed of individual spikes, resulting in a reduced amperometric spike height, slope ß, and absolute value of slope γ. The modulatory effects were independent of the PKG signal and superoxide production. This is the first study to demonstrate that 8-nitro-cGMP modulates exocytosis and provide insights into a novel regulatory mechanism of exocytosis.

Nitrosative stress in patients with asthma-chronic obstructive pulmonary disease overlap.

BACKGROUND: Asthma-chronic obstructive pulmonary disease overlap (ACO) has frequent exacerbations and a poor quality of life and prognosis compared with those of chronic obstructive pulmonary disease alone. However, the pathogenesis of ACO has not been fully elucidated yet. OBJECTIVES: The aim of this study was to investigate nitrosative stress, which causes a redox imbalance and tissue inflammation in the airways of patients with ACO, and to evaluate the relationship between nitrosative stress and the clinical course in study subjects. METHODS: Thirty healthy subjects and 56 asthmatic patients participated in this study. The asthmatic patients were divided into 33 asthmatic patients and 23 patients with ACO. The study subjects had been followed prospectively for 2 years to evaluate the clinical course. Nitrosative stress was evaluated based on the production of 3-nitrotyrosine (3-NT) in sputum cells. RESULTS: Production of 3-NT was significantly enhanced in patients with ACO compared with that in asthmatic patients. Amounts of reactive persulfides and polysulfides, newly identified powerful antioxidants, were significantly decreased in the ACO group. Baseline levels of 3-NT were significantly correlated with the frequency of exacerbations and decrease in FEV1 adjusted by age, smoking history, and blood eosinophil count. The 3-NT-positive cells were also significantly correlated with amounts of proinflammatory chemokines and cytokines. CONCLUSIONS: These findings suggested that greater nitrosative stress occurred in the airways of patients with ACO, and the degree of nitrosative stress was correlated with an impairment in the clinical course. Nitrosative stress might be related to the pathogenesis of ACO.